4. - cosmobio.co.jp · 4 respiration, they are predicted to better simulate xenob high 5 0.1 1000...

Post on 25-Jul-2020

1 views 0 download

Transcript of 4. - cosmobio.co.jp · 4 respiration, they are predicted to better simulate xenob high 5 0.1 1000...

ABSTRACT: Hepatocytes in the liver consume much oxygen for

metabolism; thus, in culturing environments with improved

respiration, they are predicted to better simulate xenobiotic

responses of the living tissue and provide an ideal system for in

vitro cytotoxicity analyses. HepG2/C3A cultured in our

gas-permeable membrane plates spontaneously formed 3D cell

aggregates (3Ds) with 5 or more cells’ height, while those in an

ordinary impermeable membrane plates formed essentially flat

2D cell aggregates (2Ds). Cell and nuclear sizes for 3Ds were

significantly smaller than those of 2Ds, reflecting near natural

dimensions. TMRM probe signal intensities measured by a

high-content imaging platform were significantly greater

allowing wider analytical ranges for 3Ds than 2Ds. The cells’

responses to FCCP and rotenone were evaluated, and individual

plane image analyses were successfully performed in 3Ds. Z’

values generated from the dose response data were typically

between 0.4 and 0.7, indicating an analytical robustness. In sum,

3D cell aggregates cultured in the gas-permeable membrane

plates can present an ideal cell culture model showing certain

physiologically-relevant cell properties for cytotoxicity HCAs.

Island-shaped 3D Cell Aggregates

on Gas-permeable Membrane Like Islands in the Ocean

33DD RREESSPPIIRRAATTIIOONN CCUULLTTUURREESS FFOORR TTOOXXIICCIITTYY HHCCAA

Jun Kanamune1, Hideki Matsunaga2, Chongmun Kim,2 Kazuyuki Yokokawa2

1 Department of Organ Reconstruction, Field of Clinical Application, Institute for Frontier Medical

Sciences, Kyoto University, Kyoto; 2 Bioscience Department, IKKO-ZU Corporation, Tokyo, Japan

60 48 36 24 12 0 m

Z stack

X-Y image

PDMS membrane

Air diffusing from underneath

Island- shaped 3D structure

11..

Culture Island 3D Cell Aggregates.

Quick & Easy

22..

Can Get 3Ds in 2 Days. Tall (Z height) & Compact

37ºC, 5% CO2

Stop dissociation with cold media

Dissociate cells by a trypsin -free agent

Cell suspension

sieved through 40 m mesh

Centrifuge once 50 x g

Seeded into membrane plates

Left undisturbed

to have cells setted at bottom

Incubated

4-10ºC

4ºC, 2 min

RT, 5 min

RT, 20 min

RT

RT

Porcine Atelo-Collagen

0

5

10

15

20

25

30

35

40

0 20 40 60 80 100 120 140

0

5

10

15

20

25

30

35

40

Width (m) 0 20 40 60 80 100 120 140

Rat-tail Collagen I

110 0

2

4

6

8

10

12

14

16

10 30 50 70 90

Porcine Atelo-Collagen

Nu

mb

er

of

3D

str

uc

ture

s

Rat-tail Collagen I

0

2

4

6

8

10

12

14

16

10 30 50 70 90 110 Peak Height (m)

33..

20

60

90

80

70

Nu

cle

i s

ize

(

m2)

50

40

30

10

0 Monolayer Island 3Ds

20 m

Island 3Ds Give Robust Dose Response Analyses. Wide Emission Range & High Z’

44..

55..

Island 3Ds Give Good Image Acquisition. Less Light Absorption & Good Probe Diffusion

Day 0 Day 0

100 m

50 m

Day 2

50 m

1000 100 10 1 0.1

5

4

3

2

1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Neg-6 Neg-5 Neg-4 Neg-3 Neg-2

Z'

Concentration ranges

Island 3Ds’ Applications Simulate Liver Zonation

& Override Crabtree Effect

PDMS

membrane

Well

Sealing film Bare partially

Bare entirely

★ For inquiry, contact: Jun Kanamune, Ph. D., E-mail:

jkanamune@frontier.kyoto-u.ac.jp or

Chongmun Kim, E-mail: kim@bioscience-ik.com

For information on VeCell gas-permeable membrane plate⇒

http://www.ikko-zu.com/bioscience/ For Acappella Analysis:

Ryo Shioda, Ph. D., Senior Application Specialist, Imaging Team,

PerkinElmer Japan, E-mail: ryo.shioda@perkinelmer.com

Gas-permeable Membrane

Stratified 3Ds Island 3Ds Spheroid

Excitation light

Emission Light

Probe entering

Detection limit Detection limit

NUCLEI NUCLEI CELLS CELLS

1 2 3 4

Sealed Bare partially

Acetamonophen (mM)

TM

RM

In

ten

sit

y

0

10

20

30

40

50

1.2

5

10 5 2.5 20

Edge line

Bare eintirely

Bare partially

Island 3Ds

TM

RM

Em

issio

n I

nte

nsit

y

0

5

10

15

20

25

30

35

40

45

0.001 0.01 0.1 1 10 100 1000

5 mM Glucose

ID50 = 0.12 M

30

0

5

10

15

20

25

0.001 0.01 0.1 1 10 100 1000

24 mM Glucose

ID50 = 10 M

Rotenone (M), 24 hrs

0

5

10

15

20

25

30

35

0.001 0.01 0.1 1 10 100 1000

0 mM Glucose

ID50 = 0.3 M

Monolayer

0

4

8

12

16

20

24

28

32

36

0.001 0.01 0.1 1 10 100 1000

24 mM Glucose

ID50 = ? M

0

5

10

15

20

25

30

35

40

0.001 0.01 0.1 1 10 100 1000

0 mM Glucose

ID50 = 0.3 M?

0.001 0.01 0.1 1 10 100 1000 0

5

10

15

20

25

30

35

40

5 mM Glucose

ID50 = ? M

Island 3Ds

50 m

Monolayer

66..

Detachment of dead cells

0

1

2

3

4

5

6

7

8

9

10

0.01 0.1 1 10 100 1000

TM

RM

Inte

nsity

FCCP (M)

0

0.5

1

1.5

0.01 0.1 1 10 100 1000

TM

RM

In

ten

sity

FCCP (M)

-30

-25

-20

-15

-10

-5

01000 100 10 1 0.1

Z'

FCCP concentration (M)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1000 100 10 1 0.1

Z'

FCCP concentration (M)